The aim of the proposed research is to gain a clearer understanding of the mechanisms by which substrates and products of mitochondrial metabolism are transported across the mitochondrial membrane. Most of these metabolites are anions in the environment of the cell but appear to accumulate in the mitochondrial matrix as a function of the delta pH across the membrane, implying that they are transported as free acids. Other carriers, which catalyze an exchange of a substrate for a product of mitochondrial metabolism (e.g. the exchange of ADP for ATP and the exchange of glutamate for aspartate) are facilitated in the physiological direction by the presence of an electrical potential gradient across the mitochondrial membrane. Careful kinetic analysis of the carriers which respond to a membrane potential is proposed as a means of distinguishing whether transport phenomenon are truly electrogenic or whether the membrane potential produces a structural change in the carriers which affects binding constants for substrates in an asymmetric manner. Kinetic analyses as a function of pH are also planned for those carriers which appear to interact directly with protons in order to learn whether interaction with the proton changes the mobility (Vmax) of the carrier or the Km for substrate. Other studies are planned to determine the effect on rates of mitochondrial transport of high ratios of membrane cholesterol/phospholipid which are likely to exist under certain pathological conditions. The studies may be of value in the interpretation of metabolic changes which occur during local tissue ischemia, since some of these changes may result from alterations of the delta pH and electrical potential gradients across the mitochondrial membranes. Likewise, the results may give a new perspective to the study of metabolic changes occurring in general metabolic acidosis.